Gene therapy is expected to become a common medical practice in years to come. Several vector systems are being developed to affect gene transfer to correct inherited or acquired diseases, such as cancer and AIDS. Lentiviral vectors are specially well suited for gene transfer into non-dividing as well as dividing cells, including stem cells. Exploiting our considerable expertise in human lentivirus (HIV-1 and HIV-2) gene regulation, we have designed novel HIV-2 vectors which have certain advantages over previously designed HIV-1 vectors (J Gen Virol, 2001). To make an informed choice, we undertook a comparative study of the packaging efficiency, gene transfer ability, and biological safety of HIV-1 and HIV-2 vectors. We find that while HIV-1 is promiscuous in packaging both HIV-1 and HIV-2 vector RNA (and possibly irrelevant RNA), HIV-2 is more specific and discriminates between self and non-self. This property likely will yield safer vectors derived from HIV-2 than HIV-1. To increase safety further and minimize recombination, we are creating chimeric vectors by DNA shuffling of HIV-1 and HIV-2 sequences. We are evaluating the efficacy of gene transfer by lentiviral vectors by using `real' genes, and not just marker genes as test models. The model genes have been chosen to represent different disease entitities, target cell specificities, and animal models. They include the Bax gene for apoptosis in neuroblastoma, the viral Vpr gene for cell cycle arrest in neoplasia, the AADC gene in Parkinson's disease to target a biochemical pathway defect, and the alpha-galactosidase (AGA) gene in Fabry disease to correct an in-born error of metabolism. Because the expression of the Bax transgene needs to be regulated not only in the host cell but also in the vector producing cell line, we are creating Tet-regulated lentiviral vectors. The use of conventional vector system yielded a poor Bax vector titer even when the packaging cell line was transduced with Bcl2 or BclXL genes. We surmise that in the context of the lentiviral vector, Bcl2 or BclXL was unable to overcome apoptosis by Bax. Alternatively, Bax may downregulate components of the vector itself. For upstream targeting and to gain better understanding of the Vpr mediated cell cycle arrest, we have undertaken microarray analysis of the genes modulated by Vpr gene transduction. For Parkinson's disease, a deficiency of L-dopamine biosynthesis, our model explores the premise that administration of high doses of L-dopa (a prodrug) with attendant side effects can be reduced if a mechanism (e.g., AADC) is provided to the neuronal cells to efficiently convert L-Dopa into dopamine. Indeed, lentiviral vector mediated AADC gene transfer into neuronal cells imparted on them the ability to affect prodrug to drug conversion. Direct injection of AADC lentiviral vector into the brain of the rat with chemically-induced Parkinson's symptoms ameliorated functional deficit (manuscript in preparation). For the Fabry disease model, transduction of fibroblasts from Fabry patients with AGA vector results in rapid clearance of the glycolipid deposits (manuscript submitted). The protocol for ex vivo transduction of mouse bone marrow cells from the Fabry knock out mouse is being optimized for subsequent transplantation. We are also exploring lentiviral vectors as tools for effective antigen delivery. With HIV infection as a model, we have created replication defective proviruses that effectively transduce dendritic cells ex vivo and likely also in vivo. Intradermal application of replication defective proviral DNA:PEI complex in mouse and monkey or infusion with genetically modified dendritic cells induces robust HIV-specific CTL immune response, surprisingly without significant antibody response (J Virol, 2001). Whether inclusion of such adjuvants as cytokines - as genes or proteins - will add on antibody response remains to be seen. Manipulation of the composition of genes in the vector and their configuration itself may also broaden the immune response. In addition to preventive vaccines, to enhance safety of these vectors for therapeutic vaccination, vector DNAs with multiple disabling mutations in a judicious mix of functional genes and regulatory elements have been designed. Some of these have been produced and are being tested for immunogenicity.